Ubiquitination is a reversible process which involves a family of deubiquitinating enzymes (DUBs) that regulate a variety of cellular processes by deconjugating ubiquitin from the substrate. DUBs are encoded by approximately 100 human genes and are classified into six families, with the largest family being the ubiquitin-specific proteases (USPs) that has more than 50 members. DUBs and their substrate proteins are often deregulated in cancers, supporting the hypothesis that targeting specific DUB family members may result in antitumor activity through enhancing the ubiquitination and subsequent degradation of oncogenic substrates and the activity of other key proteins involved in tumor growth, survival, differentiation and maintenance of the tumor microenvironment. (Hussain, S., et. al., “DUBs and cancer: The role of deubiquitinating enzymes as oncogenes, non-oncogenes and tumor suppressors.” Cell Cycle 8, 1688-1697 (2009))
USP1 is a cysteine isopeptidase of the USP subfamily of DUBs. (Nijman, S. M. B., et al. “The deubiquitinating enzyme USP1 regulates the fanconi anemia pathway. Mol. Cell 17, 331-339 (2005)) Full-length human USP1 is composed of 785-amino acids, including a catalytic triad composed of Cys90, His593 and Asp751. (Villamil, M. A., et al., “Serine phosphorylation is critical for the activation of ubiquitin-specific protease 1 and its interaction with WD40-repeat protein UAF1.” Biochem. 51, 9112-9113 (2012)) USP1 is relatively inactive on its own and full enzymatic activity is achieved only when bound in a heterodimeric complex with UAF1, a cofactor which also binds to and regulates the activity of USP12 and USP46. (Cohn, M. A., et al., “A UAF1-Containing Multisubunit Protein Complex Regulates the Fanconi Anemia Pathway.” Mol. Cell 28, 786-797 (2007))
USP1 deubiquitinates a variety of cellular targets involved in different processes related to cancer. For example, USP1 deubiquitinates PCNA (proliferating cell nuclear antigen), a key protein in translesion synthesis (TLS), and FANCD2 (Fanconi anemia group complementation group D2, a key protein in the Fanconi anemia (FA) pathway. (Nijman, S. M. B. et al. “The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway.” Mol. Cell 17, 331-339 (2005); Huang, T. T. et al., “Regulation of monoubiquitinated PCNA by DUB autocleavage.” Nat. Cell Biol. 8, 339-347 (2006)) These DNA damage response (DDR) pathways are essential for repair of DNA damage induced by DNA cross-linking agents such as cisplatin, mitomycin C, diepoxybutane, ionizing radiation and ultraviolet radiation.
In vivo studies in mouse embryonic fibroblasts (MEFs) from USP1-deficient mice show increased levels of Ub-PCNA and Ub-FANCD2 in chromatin, demonstrate impaired FANCD2 foci assembly and are defective in homologous recombination repair. Disruption of the USP1 gene in chicken cells (DT40) has been shown to result in DNA crosslinker hypersensitivity. (Oestergaard, V. H. et al. Deubiquitination of FANCD2 Is Required for DNA Crosslink Repair. Mol. Cell 28, 798-809 (2007)) Moreover, depletion of USP1 in human cell lines by siRNA results in elevated Ub-PCNA levels with increased recruitment of DNA polymerases specialized for translesion synthesis. (Cohn, M. A. et al., “A UAF1-Containing Multisubunit Protein Complex Regulates the Fanconi Anemia Pathway.” Mol. Cell 28, 786-797 (2007); Huang, T. T. et al., “Regulation of monoubiquitinated PCNA by DUB autocleavage.” Nat. Cell Biol. 8, 339-347 (2006))
In addition to regulating protein dynamics in DDR pathways, USP1 promotes tumor stem cell maintenance and radioresistance in glioblastoma via stabilization of ID1 and CHEK1 and plays a role in regulating proliferation and differentiation through deubiquitinating and stabilizing inhibitors of DNA binding (IDs) that antagonize basic helix-loop-helix (bHLH) transcription factors. (Lee, J.-K. et al., “USP1 targeting impedes GBM growth by inhibiting stem cell maintenance and radioresistance.” Neuro. Oncol. 1-11 (2015). doi: 10.1093/neuonc/nov091) shRNA knockdown of USP1 in U2OS cells induces cell cycle arrest via ID proteins and shRNA knockdown of USP1 in 143B human osteosarcoma xenografts inhibits tumor growth result. (Williams, S. A. et al., “USP1 deubiquitinates ID proteins to preserve a mesenchymal stem cell program in osteosarcoma.” Cell 146, 918-930 (2011))
Inhibition of USP1 with small molecule inhibitors therefore has the potential to be a treatment for cancers and other disorders. For this reason, there remains a considerable need for potent small molecule inhibitors of USP1.